Mechanistic synergy of hair growth promotion by the Avicennia marina extract and its active constituent (avicequinone C) in dermal papilla cells isolated from androgenic alopecia patients

Androgenic alopecia (AGA) is associated with an increased production of 5α-dihydrotestosterone (DHT) by steroid-5α-reductase (5α-R). Crude extracts from Avicennia marina (AM) and its active constituent, avicequinone C (AC), can inhibit 5α-R. We have, herein, explored the potential use of the AM extract and of AC as anti-AGA agents. To this end, we employed human dermal papilla cells (DPCs) isolated from AGA patients’ hair that express 5α-R type-1 as well as the androgenic receptor (AR) at high levels. Our in vitro experiments revealed that the AM extract (10 μg/mL) and the AC (10 μM) exhibit multiple actions that interfere with the mechanism that causes AGA. Beside acting as 5α-R inhibitors, both preparations were able to inhibit either the DHT-AR complex formation or its translocation from the cytoplasm into the nucleus (the site of DHT’s action). The treatments also increased the gene expression of growth factors in DPCs; these factors play important roles in the angiogenesis associated with hair growth. Moreover, the AM extract suppressed the apoptotic pathway, thereby postponing the initiation of the catagen phase. Taken together, our findings suggest that the AM extract and the AC could serve as natural sources for hair growth promotion and AGA treatment.


Introduction
Scalp hair follicle undergoes cycles in order to produce new hairs throughout life. It is controlled by numerous factors such as genetics, hormones, cytokines, and a number of growth studies have shown that the crude extract of AM as well as its active constituent (avicequinone C; AC) can inhibit 5α-R [27,28]. However, there has been no information regarding other actions of AM and AC that might possibly interfere with the biological processes causing AGA. Therefore, the aim of this study was to investigate the effects of both preparations on the expression of various hair growth factors (such as IGF-1, KGF, HGF, and VEGF), and their ability to inhibit the androgenic action of DHT through an interference at the levels of the DHT-AR complex formation or its translocation from the cytoplasm into the nucleus of the DPCs.

Plant material and active compound
The heartwood powder of AM was purchased from Chao Krom Poe Dispensary; a local Thai-Chinese medicinal store (Bangkok, Thailand) in June 2019 (Lot No. 190619). The raw material was determined to have an extractive value of 7.0%w/w using 95% ethanol and moisture content of 9.5%w/w by thermogravimetric method. The raw material was authenticated by comparing its TLC-chemical fingerprint of the ethanol extract with that reported previously [28]. AC was obtained by chemical synthesis, according to a method previously described in detail [29].

Preparation of the AM extract and quality assessments
The AM raw material was first ground into powder, followed by an ethanol extraction using the method as described previously [28]. Practically, the extraction was conducted by maceration (200 g/L of 95% ethanol) for 24 h at room temperature for three cycles. The ethanolic extracts were pooled and evaporated until dryness at 40˚C using a rotary evaporator. The quality assessment of the obtained AM extract was done by TLC which showed the chemical profile of the AM extract with AC used as a standard marker (S1A Fig in S1 File). The TLC system and conditions used in the analysis has been described in the report published previously [28]. The AM extract appeared to be relatively stable upon storage for at least three years at -20˚C as shown by the same TLC analysis. Only slightly decrease in the intensity of the AC band was observed (S1A Fig in S1 File). Quantitatively, a standard curve of AC marker (working range: 100-1000 ng with correlation coefficient (r 2 ) = 0.99) was constructed based on TLC densitometric method using the wavelength of 254 nm (S1B Fig in S1 File). The results showed that the AM extract freshly prepared in June 2019 contained the AC content of 6.2% dry weight compared with 5.1% AC of the same AM extract stored for three years under -20˚C. The structure of AC in the AM extract was identified as avicequinone C based on the spectral data of both 1H-NMR and 13C-NMR reported previously [28]. In this study, the stock solutions of the AM extract (1 mg/mL) and of the AC compound (1 mM) were prepared by using DMSO and kept at 4˚C before use.

Isolation of DPCs from human hair follicles, and cell culture establishment
DPCs were isolated from the collected hair samples by micro-dissection, and were cultured by using the method described previously [30,31] with slight modification. Briefly, each hair follicle was dissected under the stereomicroscope at the hair root area of the dermal papilla. The pear-shape structure of the dermal papilla was extruded out by applying gentle pressure, and was then transferred and scratched onto a culture dish containing complete DMEM supplemented with 10% FBS and 1% of an antibiotic-antimycotic. The dermal papilla was incubated at 37˚C under a humidified atmosphere of 95% air and 5% CO 2 for 2 to 4 weeks, or until the dermal papilla was attached. Once the first cell migration became apparent, the medium was thereafter changed twice per week [32]. Once the cells reached full confluency, they were subcultured by using 0.25% trypsin/EDTA. The DPCs in their third to fifth passages were used in our experiments.

Detection of the 5α-R and of the androgen receptor in primary DPC cultures
RT-PCR was used in order to check for the expression of target genes in primary DPCs, including those of 5α-R1, 5α-R2, and AR. The total RNA from DPCs (1 × 10 5 cells/well in 6-well plates) were extracted by using the GENEzol TM reagent according to the manufacturer's instruction. cDNA was synthesized by using the revert-aid premium reverse transcriptase. The forward and reverse primers used for gene amplification are listed in Table 1. The PCR products were analyzed by using 1% agarose gel electrophoresis.

Cytotoxicity assay
The optimal non-toxic concentrations of the AM extract and of the AC used in this study were determined by using the PrestoBlue1 reagent (Invitrogen, USA). DPCs at a density of 5 × 10 3 cells/well in 96-well plates, were cultured overnight in complete DMEM. The cells were separately treated with various concentrations of the AM extract (0-40 μg/mL) and of the AC (0-40 μM). Cell viability was measured after an incubation with PrestoBlue1 in Roswell Park Memorial Institute (RPMI) medium (Invitrogen, USA) at 37˚C, for 48 h. If there were viable cells, PrestoBlue1 would change from a blue to a purple-pink color, that could be spectrophotometrically detected at 570 nm.

Determination of the 5α-R inhibitory activity
The 5α-R inhibitory activity of AM and AC was determined by using a combination of a cell (DPC)-based assay and high-performance TLC detection, as described previously [28]. Briefly, the DPCs at a density of 2 × 10 5 cells/mL were seeded into 6-well plates, and were treated separately with 1 mL of 30 μM T and 1 mL of 0.1% DMSO (internal control), 1 mL of 30 μM T and 1 mL of 10 μg/mL of AM, 1 mL of 30 μM T and 1 mL of 10 μM of AC, and 2 mL of 0.1% DMSO for the negative control. After a 36-h incubation, the culture medium was collected and the attached cells were tested for cell viability by using 1× PrestoBlue1 reagent in RPMI medium. T and its product, DHT, were extracted from the cell culture medium by using an equal volume of ethyl acetate. The ethyl acetate fraction was dried and reconstituted with 20 μL of methanol, prior to spotting on a TLC silica gel 60 F254 aluminum plate. The TLC plate was developed by using toluene, acetone, and acetic acid at a ratio of 8:2:0.2 as the mobile phase. The developed TLC plate was then dipped in a solution of 42.5% phosphoric acid, and was heated at 120˚C for 20 min, thereby allowing for the visual detection of DHT at 366 nm by using a TLC Reprostar imager. The amount of DHT was quantified by using an image analyzing program. The inhibitory activity was determined through the decrease in the DHT production observed relatively to the internal control.

Cell cycle analysis
DPCs at density of 2 × 10 5 cells/mL were seeded into 6-well plates, were further cultivated for 24 h, and were treated separately with the AM extract (10 μg/mL) and the AC (10 μM) for 48 h. After 48 h of incubation, the cells were harvested and centrifuged for 5 min at 1,200 rpm. Subsequently, the supernatant was removed, and the cells were resuspended in 0.5 mL of PBS. We added 4.5 mL of 70% ethanol in tubes, and kept them on ice. Subsequently, 0.5 mL of the cell suspension was transferred into the tubes containing the cold 70% ethanol fixative, and the cells were kept in the fixative for time �2 h, on ice. The ethanol-suspended cells were then centrifuged for 5 min at 1,200 rpm, in thoroughly decanted ethanol. The cells were washed three times with PBS by resuspending the pellet in 2 mL of PBS, and by centrifuging again in order to remove the supernatant. The cells were resuspended in PBS containing RNase A at 100 μg/ mL, and were incubated at 37˚C for 30 min. A propidium iodide (PI) solution was added to a final concentration of 50 μg/mL, and the mixture was then incubated at room temperature for 30 min (while protected from light). Samples were analyzed for their DNA content by a FAC-Sort flow cytometer (Becton Dickinson, Rutherford, NJ, USA), with the excitation set at 488  nm and the emission filter set at 600 nm. The CellQuestTM Pro software (Becton Dickinson) was used for the identification of the sub G0/G1 phase DNA distribution in the apoptosis process. Ten thousand cells in each sample were analyzed and expressed as a percentage of total cells.

Determination of the gene expression of hair-growth factors
The expression of four targeted hair-growth factors (namely, IGF-1, HGF, VEGF, and KGF) was determined by RT-PCR (transcriptional level). The primers were designed by using the NCBI primer design tool from the full-length mRNA sequences obtained from the NCBI Gen-Bank, and were made to order by 1 st Base Laboratories (Selangor, Malaysia). The forward and reverse primers for amplification are listed in Table 2. DPCs at a density of 2 × 10 5 cells/mL were seeded in a culture dish for 24 h, followed by the addition of various concentrations of the AM extract and of the AC, and an incubation for 24 and 48 h. The total RNA from each treatment was then extracted from the cells by using the GENEzolTM reagent. The PCR products were run on a 1% agarose gel electrophoresis, were visualized by ethidium bromide staining, were de-stained with water, and were visualized under a UV trans-illuminator by using a gel documentation system.

Effect of the AM extract on the protein expression of hair-growth factors
DPCs at a density of 2 × 10 5 cells/well were seeded into 6-well plates for 24 h, and were cultured in the presence of the AM extract (10 μg/mL) for 48 h. After washing with PBS, the lysates of the treated cells were prepared by incubating the cells for 30 min in ice-cold lysis buffer containing 20 mM Tris�HCl (pH 7.5), 0.5% Triton X, 10% glycerol, 150 mM sodium chloride, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 100 mM phenylmethylsulfonyl fluoride, and a commercially available protease inhibitor cocktail (Roche Molecular Biochemicals). The cell lysates were centrifuged at 12,000 rpm, at 4˚C, for 15 min, and the supernatant was collected and determined for its total protein content by the BCA protein assay kit (Bio-Rad, Hercules, CA). An equal amount of 50 μg of protein from each sample was boiled in Laemmli loading buffer at 95˚C, for 5 min, so as to induce protein denaturation. The proteins were subsequently loaded on 10% SDS-polyacrylamide electrophoresis gels. After separation, the proteins were transferred onto 0.45 μm nitrocellulose membranes (Bio-Rad, Hercules, CA). Following a blocking with 5% nonfat milk in TBST (25 mM Tris�HCl (pH 7.5), 0.05% Tween-20, and 125 mM NaCl) for 2 h, the membranes were incubated with the appropriate primary antibodies against Bcl-2, Bax, caspase-3, VEGF, KGF, HGF, and BMP-4, for 10 h, at 4˚C. The resulting membranes were then washed three times  with TBST (for 15 min), and were incubated with horseradish peroxidase-coupled secondary antibodies for 1 h, at room temperature. The signals from the target proteins were detected by the use of a chemiluminescence substrate (Super Signal West Pico, Pierce, Rockford, IL). The intensity of the protein signal was quantified by using unsaturated images on the ImageJ 1.53e software.

Determination of the inhibitory effect on the DHT-AR complex formation and translocation from the cytoplasm to the nucleus
In this study, the immunofluorescence staining of the AR was used in order to determine the possible effect of the AM extract and of the AC on the DHT-AR complex formation and translocation from the cytoplasm to the nucleus of DPCs. This was performed by using the method previous described [33]. Practically, DPCs were first seeded into 24 wells-plate, and were cultured for cell attachment for 24 h. The cells were then treated separately with the AM extract (10 μg/mL) or the AC (10 μM) in the presence of 30 μM of DHT. The positive control was incubated with 30 μM of DHT and 20 nM of flutamide (an androgen-AR binding inhibitor). After the treatment, the cells were washed three times in ice-cold PBS, and were fixed with absolute methanol for 15 min at 20˚C. After fixation, the cells were washed three times in icecold PBS, and were incubated with 2% (v/v) of BSA in PBS-T (PBS with 0.5% TritonX-100; nonionic detergent) for 1 h in order to permealize and block the cells, so as to allow the antibody to access intra-organelle antigens and to block nonspecific antiserum binding. This step was followed by an overnight incubation (at 4˚C with a monoclonal rabbit anti-AR antibody diluted in blocking buffer. After washing off the primary antibody with PBS-T, the cells were incubated (for 4 h in the dark) with a secondary Alexa Flur488-labeled donkey anti-rabbit IgG antibody (1:600; Molecular Probes) diluted in blocking buffer. After removing the secondary antibody, the cells were washed three times with PBS-T, and their nuclei were counterstained with 1 μg/mL of Hoechst for 15 min. The cells were imaged by using fluorescence spectroscopy at room temperature.

Statistical analysis
All data were obtained from three individual experiments. The obtained data are presented as mean ± SD, and were analyzed by using one-way analysis of variance (ANOVA) via GraphPad Prism 9.0. Statistical significance was considered for p values lower than 0.05.

Isolation of DPCs and expression of target genes
DPCs were isolated by a simple micro-dissection from the bulb of the dissected hair follicles obtained from the occipital scalp region of a patient undergoing hair transplant. The DPCs with membrane-bound, pear-shaped structures were isolated from an intact hair follicle (Fig  1Aa and 1Ab). Structurally, the DPCs are enveloped with membrane-bound, pear-shaped structures; we, thus, needed to scratch the membrane part of the DPCs in order to allow for the whole pear-shaped structure to come out and stay at the bottom of the culture dish, and then open the explant and leave it untouched until the migration of the initiated cells from the explant; a migration that was observed after 14-30 days [34]. Fig 1Ac-1Ae) show the migration of the cells from the explant in the form of a spread-out growth, until they reach full confluency. As previously reported [33][34][35][36][37][38][39][40], the isolated DPCs appeared to have specific characteristics, including a flattened morphology, an irregular spindle shape, and an ability to form multi-layered aggregations at full cell confluency (Fig 1Af-1Ah).
Since the action of androgens causing AGA is known to be exerted through the functions of 5α-R and the AR, the gene expression of the two targets in the DPCs was a prerequisite for studying the mechanistic effects of AM and AC as anti-AGA agents. These gene expressions were determined by RT-PCR by using specific primers for the two 5α-R isoforms (5α-R1 and 5α-R2) and the AR protein. The results revealed that both the AR and 5α-R1 were strongly expressed in primary DPCs, along with a much lower expression of the 5α-R2 (Fig 1B). Similar results have been previously reported regarding the AR and 5α-R1 mRNAs expressed in all parts of the hair follicle (including the DPCs), while the 5α-R2 mRNA has been previously shown to be expressed only in the mesenchymal parts of the dermal papilla and in the connective tissue sheath [41]. The same report has also shown that the intensity of the expression of these genes in each part of the hair follicle did not differ between follicles from balding and those from nonbalding scalps [41]. As far as the AR expression is concerned, it has been demonstrated by hormone binding assays and RT-PCR that the AR expression is significantly higher in bald DPCs than in nonbald ones [42]. Thus, the strong expression of 5α-R1 ( Fig  1Ba) and AR (Fig 1Bb) in our primary DPCs can be used as a model for investigating the mechanism of action of the AM extract and of AC.

The effect of the AM extract and of AC on the cell cycle of isolated primary DPCs
Hair follicle morphogenesis is characterized by a tightly regulated balance of cell proliferation, differentiation, and apoptosis [43]. In order to observe the effects of AM and AC on the cell cycle of isolated DPCs, the primary cells were first determined for the cytotoxicity of both the extract and active compound by using MTT assay for 48 hr. This incubation time was optimized based on the time-course study of 5α-R enzyme activity assay (0, 6, 12, 24, 36 and 48 h) using the primary DPCs ( S6 Fig in S1 File). The results showed that the treatment of AM at the concentration range of 2.5-20 μg/mL and AC at 2.5-20 μM did not cause significant cytotoxicity on DPCs, compared with the non-treated cells (Fig 2). At the maximum non-toxic concentrations of 20 μg/mL AM and 20 μM AC, the cell viability was observed to be in the range of 80-85% which is lower than the acceptable 90% cell viability. Therefore, we selected the concentrations of 10 μg/mL AM and 10 μM AC for cell cycle analysis and further experiments. DPCs were treated with AM (10 μg/mL) and AC (10 μM) for 48 h, followed by an analysis of their cell phase distribution by a flow cytometer. The results showed that both the AM extract and the AC significantly decreased the number of cells in the G0 phase, and simultaneously increased those in the G2-phase (as compared to the control; Fig 3). On the other hand, no significant differences were observed between the treated and the non-treated cells in terms of the cell population numbers undergoing their G1 and S phases. Since the G0 cells represent apoptotic cells and the G2 phase represents the active phases of DNA synthesis and cell  proliferation, the obtained results suggest that AM and AC not only prevent the apoptosis of DPCs, but also induce the proliferation of these cells.

The effect of the AM extract and of AC on the inhibition of 5α-R
By using the DPC-based assay coupled with a non-radioactive thin layer chromatography (TLC) for determining the 5α-R activity [28], both the AM extract and the AC were found to clearly inhibit 5α-R (Fig 4), Quantitatively, the AM extract at 10 μg/mL exhibited a 73.40% (±8.40%) inhibition (lane 3), while AC at 10 μM (or 2.56 μg/mL) exhibited a 67.71% (±3.81%) inhibition (lane 4). Dutasteride (DU; a competitive inhibitor of 5α-R used as a positive control at 1 μM) exerted a 100% inhibition of 5α-R (lane 2). The obtained results suggest that both the AM extract and the AC could decrease the 5α-R activity, and cause a reduction of the DHT production by more than 50% in DPCs. Moreover, the AM extract (at 10 μg/mL) exhibited the strongest 5α-R inhibitory activity compared to other potential extracts and pure compounds (S2 Fig in S1 File); notably, with a similar level of potency to the commercial inhibitor of finasteride (1μM). This may reflect the synergistic effects of the constituents present in the AM extract [29]. To prove this, however, it is necessary to find other active constituents in AM, followed by observing the 5α-R inhibitory activity of their combinations with AC.

The effect of the AM extract and of AC on the inhibition of the DHT-AR complex formation and of its nuclear translocation
AGA is an androgen-mediated disorder the mechanism of which involves the nuclear translocation of the AR upon the binding of androgens, thereby resulting in the activation their  Table. All data were obtained from experiments performed in triplicate, and are presented as mean ± SD (n = 3). ***p<0.005 versus non-treated cells. androgenic activity. Therefore, the effects of the AM extract and of AC on the nuclear translocation of AR in DPCs were investigated. In this study, DHT (at 30 μM) was used as the active androgen for stimulating its binding with AR and for causing the nuclear translocation of the DHT-AR complex. Flutamide was used as positive control in this study. Flutamide is a selective antagonist of the AR, where it competes with the androgens T and DHT for its binding to the AR in various target tissues [35]. The results of the nuclear translocation were obtained by using the immunofluorescent staining of AR and the blue fluorescence (Hoechst dye) staining of nuclei. As shown in Fig 5, it was found that DHT readily activated the translocation of the AR (presumably in the form of a DHT-AR complex) from the cytoplasm into the nucleus, as observed by the much higher intensity of the green fluorescence in the nucleus than in the cytoplasm (Fig 5B) when compared with the control (Fig 5A). In the presence of the active AM extract (10 μg/mL) (Fig 5D) or the AC (10 μM) (Fig 5E), it appeared that both preparations could counteract the effect of DHT by exhibiting a marked decrease in the levels of nuclear translocation of the AR (Fig 5D and 5E). Interestingly, the efficiency of the positive control (containing 10 μM flutamide) in inhibiting the nuclear translocation of the AR (Fig 5C) was found to be lower than those of the AM extract and the AC. Furthermore, the AM extract and the AC exhibited a reduction of the AR translocation after a stimulation with T at 30 μM (S3 Fig in S1 File). These results suggested that the AM extract and the AC were able to inhibit the both DHT-AR and T-AR complex formation and translocation from the cytoplasm into the nucleus (which is the site of the DHT action) in DPCs. Whether the inhibition took place at the step of the binding of the DHT to the AR or at the complex translocation step remains unclear. However, our finding is in agreement with those of previous reports showing that the extract of Scutellaria baicalensis and its active compound (baicalin) were capable of inhibiting the translocation of the AR, with implications for the prevention of AGA [33,36].

The effect of the AM extract and of AC on the gene expression of hair growth factors
As mentioned earlier, the overproduction of DHT in AGA patients alters the production of some growth factors that are secreted by DPCs (particularly, IGF-1, KGF, HGF, and VEGF); these growth factors are known to be involved in the positive regulation of the hair growth cycle [6][7][8]. In an attempt to examine whether the AM extract and the AC have any effect on the mRNA expression of these growth factors, the RT-PCR technique was used in order to quantitate their gene expression. It was found that both the AM extract and the AC could significantly increase the mRNA levels of VEGF and HGF in the presence of T (30 μM) (Fig 6A  and 6B), without affecting those of KGF and IGF-1 (S4 Fig in S1 File). The upregulation of VEGF was found to be highest at 6 h, with approximately a 3.5-fold increase (as compared to the control; Fig 6A), followed by its continuous decrease to nearly the control level at 48 h. In the case of HGF, on the other hand, its gene expression in response to a treatment with the AM extract and the AC appeared to be slower, with a 2.2-and a 4.8-fold increase of the HGF mRNA at 24 and 48 h, respectively (Fig 6B). Therefore, it seems that the induction of the gene expression of VEGF (6 h) in response to a treatment with either the AM extract or the AC, is DPCs were incubated with 30 μM of T, and were treated with the AM extract or with AC at various times before determining the gene expression of (a) VEGF and (b) HGF. The expression levels of each of these growth factors were analyzed by using the ImageJ 1.53e software. All data were performed in triplicate, and are herein presented as mean ± SD (n = 3). *p<0.05, **p<0.01, and ***p<0.005 versus non-treated cells.
https://doi.org/10.1371/journal.pone.0284853.g006 quicker than that of HGF (24 h). In the literature, the expression of VEGF in DPCs during the hair cycle has been reported to fluctuate, with a strong expression being recorded during the anagen phase, and lower expressions being recorded during the catagen and the telogen phases [18]. Similarly, the upregulation of the HGF mRNA has also been observed in the anagen phase during the DNA synthesis in keratinocytes deriving from the human hair follicle development [37]. Our results are similar to those of a previous report showing that the Geranium sibiricum extract promoted hair growth (both in vitro and in vivo) by upregulating the mRNA levelS of VEGF and HGF, and that the reduction in the expression of these genes led the cells to the catagen phase [37]. For KGF and IGF-1, the observed negative induction in their mRNA expression suggested that neither of these hair growth factors are targets of the AM extract and of the AC. Alternatively, a reason for our observations might be that the time of their response was not covered by herein adopted timeframe of study.

Effect of the AM extract on the protein expression of hair growth factors
In addition to the effect on the mRNA expression of hair growth factors, the effect on the protein expression of these genes was also examined. This was carried out by Western blotting with a focus on the hair growth factors KGF, HGF, and VEGF. In this experiment, only the AM extract was used for the cell treatment, as all results obtained above revealed similar effects between both the AM extract and the AC. The concentration of the AM extract was, again, set at 10 μg/mL; a concentration that has been shown to be non-toxic (Fig 2). Our findings revealed that the AM extract was able to significantly increase the protein expression of all three growth factors (Fig 7). In fact, it can be seen that the AM extract can significantly increase the protein levels of HGF after 12 and 24 h of incubation (Fig 7A), and those of KGF after 6, 12, and 24 h of incubation ( Fig 7B). As far as the VEGF is concerned, its protein overexpression was found to increase significantly at 6 h, followed by a rapid decline at 12 h ( Fig  7C). By comparing between the results of the mRNA expression (Fig 6) and those of the protein expression (Fig 7), both HGF and VEGF seem to exhibit a good correlation between the two expression levels in terms of their time responses to the AM extract treatment. KGF, on the other hand, was shown to be well responsive to the AM extract treatment only at the protein expression level (Fig 7B), but not at the mRNA expression level (S3a Fig in S1 File). It might be possible that the endogenous mRNA of KGF in DPCs was already available as a result of normal gene expression and, thus, the cells were able to readily respond to the AM extract treatment through the protein expression of KGF. Therefore, all results suggest that the AM extract can stimulate the production of hair growth factors such as the VEGF, the HGF, and the KGF.

Effect of the AM extract on the protein expression of anti-apoptotic mediators
Apoptosis plays a central role in the regulation of the hair follicle during the catagen phase [38]. Its effects are modulated by a complex signaling pathway involving the important controlling mechanisms of Bcl-2, Bax, and caspase-3 [38]. In an attempt to evaluate whether the AM extract (10 μg/mL) affects the hair-inducing activity of DPCs, the expression levels of these associated proteins were examined. Our results revealed that the AM extract was capable of inducing the protein expression of Bcl-2 by a significant 75% after 6 h of incubation ( Fig  8A). Simultaneously, the protein levels of Bax and caspase-3 were found to be considerably decreased after 6-24 h of incubation (Fig 8B and 8C). These results are similar to those obtained after a minoxidil treatment [18] or a treatment of DPCs with macrophage-derived extracellular vesicles [17], that promoted the migration and the proliferation of DPCs as well as the delay of the apoptotic process. Finasteride, a specific 5α-R type 2 inhibitor, has been found to affect the caspase expression in the hair follicles, thereby causing an induction of the active growth hair cycle [39]. Tetrahydroxystilbene glucoside isolated from Polygonum multiflorum has also been reported to possess an anti-hair loss activity, by suppressing Bax that induces apoptosis [40]. Therefore, our findings support the ability of the AM extract to promote hair growth by delaying the initiation of the catagen and/or the apoptotic phase in the hair cycle.

Effect of the AM extract on the protein expression of BMP-4
The BMP pathway is a key regulator of the genetic program controlling the hair shaft differentiation in the hair follicle. It has been reported that a negative effect of the BMP signaling can induce hair growth in the hair follicle that is in its anagen phase, whereas a positive effect can promote the hair growth shifting to the telogen phase [15]. Thus, the effect of the AM extract on the expression of BMP-4 in DPCs was determined. Our Western blotting analysis revealed that the AM extract was able to continuously decrease the protein levels of BMP-4 during the 24 h of the treatment (as compared to control; Fig 9). This finding suggests that the AM extract potentially induces hair growth at the telogen phase by attenuating the BMP-4 protein in DPCs. Similarly, previous studies have shown that the enhancement of the BMP signal activation with noggin (a BMP antagonist) can result into a significant delay of the induction of the hair follicle, and lead to progressive baldness [15].
Taken together, the AM extract and the AC clearly act as 5α-R inhibitors that can significantly reduce the formation of DHT in DPCs. The pure compound of AC at 10 μM exhibited DPCs were incubated with T (30 μM) as a control, and were treated with the AM extract (10 μg/mL) for various periods (6, 12, and 24 h). The bar graph data analysis of the undertaken Western blotting is presented relatively to the control group at 0 h. The expression levels of Bcl-2 (a), Bax (b), and cleaved caspase-3 (c) are presented. All data are presented as mean ± SD (n = 3). *p<0.05, and ***p<0.005 versus non-treated cells.
https://doi.org/10.1371/journal.pone.0284853.g008 its highest inhibitory activity, and caused a reduction of the DHT production by 67.7% in DPCs, whereas the AM extract at 10 μg/mL was able to reduce the formation of DHT by 73.5% in DPCs. It is well-known that androgens exert their activity through binding with the AR. The reduction of DHT would, on one hand, lead to a lowering of the level of the AR-DHT complex. On the other hand, our results also suggest that both the AM extract and the AC can interfere directly with the binding of DHT to the AR, thereby resulting in a decrease of the translocation of the AR-DHT complex from the cytoplasm to the nucleus. Moreover, the treatment with the AM extract or the AC in the presence of T revealed a significant increase in the mRNA expression of the key growth factors of VEGF and HGF. These results suggest that both the AM extract and the AC can promote hair growth by stimulating the expression of DPCs were incubated with T (30 μM) and were treated with the AM extract (10 μg/mL) for 6, 12, and 24 h. DPCs were incubated only with T (30 μM) as a control. The histograms represent the relative levels of the protein expression (relative to the control group). Quantitative analysis of the protein expression was determined by using the ImageJ software. Western blot showing the protein expression of BMP-4 in each condition, and quantitative analysis of the protein expression. The data are presented as mean ± SD (n = 3). *p<0.05, and ***p<0.005 versus non-treated cells.
https://doi.org/10.1371/journal.pone.0284853.g009 VEGF and HGF; two signal transduction molecules that regulate the hair follicle cyclic growth. Both VEGF and HGF play important roles in the angiogenesis associated with the hair growth and hair cycling [6][7][8]. In addition, this study also revealed that the levels of Bcl-2 (that protects hair follicle cells from apoptosis, thereby resulting in maintaining the hair follicle in the anagen-catagen phase) were significantly increased as a result of a treatment with the AM extract. On the other hand, the levels of Bax and caspases-3 were markedly decreased in DPCs, thereby resulting in an inhibition of cell death. Similarly, the AM extract strongly attenuated the protein levels of BMP-4, thereby potentially causing hair growth stimulation. These results suggest that the AM extract can promote hair growth by stimulating the expression of VEGF and HGF that proceed with an intercellular signal transduction, and can regulate the hair follicle cyclic growth (Fig 10).
The AM extract inhibits 5α-R (1) and reduces the nuclear translocation of AR (2). The AM extract upregulates the mRNA levels of VEGF and HGF (3). In the cell cycle pathway, the AM extract diminishes the cell population in the G0 phase, and increases the number of cells in the G2 phase (4). The AM extract reduces the expression of Bax and cleaved caspase-3, and induces the expression of Bcl so as to delay the induction of the catagen phase (5). The protein expression of hair growth factors (HGF, KGF, and VEGF) was found to increase after a treatment with the AM extract, thereby promoting the cells' retaining in the anagen phase (6). Moreover, the AM extract reduced the expression of BMP-4, thereby facilitating the cells' turning to the anagen phase (7).

Conclusions
AGA is a common type of scalp hair loss in both men and women. It is a genetically determined disorder associated with the increasing production of DHT, that is formed from T by 5α-R. Androgens and their receptor (AR) cause signaling and biochemical reactions that result in the alteration of cytokines and growth factors, thereby leading to premature hair loss. Therefore, potential drugs to be used for the prevention or the cure of AGA should be able to inhibit 5α-R in order to reduce the androgen formation or to block the AR activation in DPCs. We have shown in this study that the target DPCs could be isolated by a simple microdissection without the use of enzyme treatments. The isolated DPCs clearly exhibited the expression of target genes (especially those of the 5α-R1 and the AR), thereby convincing us that these cells are a suitable model for the study of the mechanisms of action of the AM extract and of the AC. These included their effects on the 5α-R enzymatic activity, the nuclear translocation of the DHT-AR complex, and the expression of certain growth factors. Our results suggest that the AM extract as well as its active compound (AC) possess a potent in vitro 5α-R inhibitory activity coupled with an inhibition of the androgen effect in DPCs causing the decrease of the androgen-dependent activation of the AR. Furthermore, they increase the mRNA expression of some growth factors that are generally believed to stimulate hair growth. Due to our experiments being conducted on DPCs isolated from AGA patients, these finding might support the use of the AM extract and of the AC in future hair growth / protecting strategies for AGA patients. Moreover, there is a potential for the development and future application of the AM extract as a natural drug promoting hair growth, in both cosmetics and pharmaceuticals used for the treatment of AGA.